Professional Documents
Culture Documents
The detection of HCP is usually using silver staining, western blot, threshold system, ELISA, and
LC-MS. In the early stages of protein drug development, HCP was measured using silver staining
and western blot analysis. However, there were limitations in the final drug evaluation due to
lack of reproducibility, quantification, and detection limit. Recently, threshold system, ELISA and
LC-MS are preferred.
Evaluation of HCP
The HCP antibodies are produced by immunizing suitable animal species then purify the
produced antiserum and evaluate before use. The concepts of HCP antibodies, the HCP is
injected in suitable animal, then the animal will produces the antibodies that can be collected as
HCP antibodies. The antibodies present in the antiserum are generally reported to be stable for
about 10 years at -80℃.
1. Major items
- Choosing antibody production: rabbit, guinea pig, goat or rat
- Purification method: affinity (Protein A or G), immune affinity (antigen column)
- Possibility of conjugation between antibody and enzyme.
2. Evaluation
- Specificity: SDS-PAGE, western blot. HCP reaction should be observed in western blot results
but not with the target protein. Band patterns should be similar when electrophoresis and
western blot are compared. The target protein in western blot is not visible in western blot,
however in SDS-PAGE and Silver staining the target protein (protein of interest) in line 3 and
4 still visible, because the interest protein in western blot already bind by antibody.
- Titer: measure titer using ELISA to check sensitivity.
1. Antigen
- Mock-run: running process of HCP production with the similar procedure and materials
- Selection of Antigen
- Characterization
2. Immunization
- Control of preimmune sera: the animals should have the control immune before injected with HCP
- Immunization: injected the HCP protein into animals (rabbit, goat etc)
- Control of sera: for cross reaction against product protein.
3. Qualitative of Sera
- Purification of antibodies (protein A using affinity chromatography)
- 1-D and 2-D electrophoresis and western blot.
4. Quantitative assay
C. ELISA FORMAT
1. Initial antibody and HCP range selection: determine with titer test
To develop how much range concentration that can be used with ELISA.
2. Decide initial concentration of antibody-enzyme conjugate
Aliquot a generous amount of HCP, based on the absorbance value from antibody-enzyme
conjugate serially diluted with dilution buffer to use a dilution factor of 70% of the maximum
absorbance to set the initial testing conditions. The set point is the absorbance maximum point
in dilution factor of 70% to get dilution factor test.
3. Define ELISA Assay format
Decide the format that satisfies limit of quantitation (LOQ) and recovery rate based on direct,
indirect, sandwich, competition methods.
- Direct: the HCP or protein direct to its antibody (anti-HCP) which is conjugated with the HRP
(anti-HCP-HRP conjugated)
- Indirect: the HCP or protein bind with anti-HCP, then react with second antibody which is
binding with HRP conjugated.
- Sandwich: the protein of interest is put on the center between anti-HCP and anti-HCP-HRP
conjugated. So, the plate already coating with the antibody (anti-HCP), then add with the
HCP or protein and next is react with the anti-HCP-HRP conjugate.
- Competitive: the HCP protein is reacting with the anti-HCP-HRP conjugate then adds with
HCP competition.
4. Define optimal ELISA assay conditions that satisfies LOQ and recovery rate
- Binding: microplate types, binding conditions, binding method (hydrophobic interaction, use
of Progein A/G, covalent bond, etc.,), binding buffer (10mM PBS, 50 mM carbonate, 20 mM
Tris-HCl), binding temperature and duration (37℃ for 1-3 hours, room temeperature 1-3
hours, 4℃ 16-24 hours)
- Blocking: generally, using bovine serum albumin (BSA) or best reagent selected based on
analytical method.
- Assay condition
Incubation temperature: higher temperature means greater speeds of binding, lower
temperature means better reproducibility and precision.
Incubation time: keep reaction time short if reagent concentration is high.
Mixing: if mixing, increase reaction time.
Anti-HCP antibody type and dilution factor: define optimal conditions at 500-50.000x
dilution.
Incubation reagent type: define optimal conditions with blocking reagent and reagent
mixture. Type of reagent: Protein agents (BSA, Casein, Gelatin, Nonfat dry milk, ovalbumin),
nonprotein agents (polyvinyl pyrrolidone, polyvinyl alcohol) and detergent (Tween-20, triton
X-100).
D. Threshold Format
In the Threshold assay, the conjugate is using biotinylated and streptavidin (to bind with the
protein).
1. Threshold ILA assay format selection
- Sandwich, competition format
- Sandwich (commonly used)
- Antibody type and count, antigen size, antigen epitope count
- More sensitive than ELISA
- Analysis time is short at 3 hours
- Difficult if there are a lot of analysis samples.
- Streptavidin
- Biotinylated antibody: biotin will react with streptavidin
- Fluorescent antibody
- Anti-fluorescent urease
- Reaction stage: formation of tripartite complex between HCP and two labeled antibodies
(streptavidin conjugated with biotinylated antibody, then react with the HCP, then HCP bind
with fluorescent antibody-anti fluorescent urease)
- Separation stage: after addition of streptavidin, immune complex are then captured on
biotinylated nitrocellulose membrane.
- The detection stages start with incubation of the membrane with anti-fluorescein-urease
conjugate. The urease hydrolyses added urea into ammonia that alters the pH of the
solution, then change the surface potential on membrane, and measure by silicon sensor.